The present invention relates to an apparatus, system and method for executing and analyzing biological and chemical reactions automatically. More particularly, the present invention relates to an apparatus, system and method for automating the execution of a nucleic acids reaction, such as, for example, the polymerase chain reaction (PCR) in an open sample tube, thus allowing the automated analysis thereof either during or immediately following the nucleic acid reaction.
Diagnostic and research biology and chemistry rely heavily on the ability to perform various biological and chemical reactions in vitro. Such reactions are typically accomplished under controlled conditions which, aside from appropriate sample preparation, typically include temperature and time modulation.
An excellent example to an in vitro biological reaction is the polymerase chain reaction (PCR). The methodology of the polymerase chain reaction is described in detail in U.S. Pat. Nos. 4,683,202 and 4,683,195 which are incorporated herein by reference.
PCR has proven to be a phenomenal tool for diagnostics and research in many scientific fields including, but not limited to, genetics, molecular biology, cellular biology, clinical chemistry, forensic science, and analytical biochemistry, see, for example, Erlich (ed.), 1989, PCR Technology, Stockton Press (New York); Erlich et al. (eds.), 1989, Polymerase Chain Reaction, Cold Spring Harbor Press Cold Spring Harbor, New York; Innis et al., 1990, PCR Protocols, Academic Press New York; and White et al., 1989, Trends in Genetics 5/6:185-189.
The use of PCR can replace a large fraction of molecular cloning and mutagenesis operations, commonly performed in bacteria, thus providing speed, simplicity and at the same time lowering costs. Furthermore, PCR permits the rapid and highly sensitive qualitative and even quantitative analysis of nucleic acid sequences, enabling non-radioactive associated detection, thus overcoming the risks and restrictions associated with the utilization of radioactive isotopes.
Additional reactions which are propagated by carefully controlling and cycling the reaction temperature, include, but are not limited to, chemical amplification of nucleic acid sequences, as for example described in U.S. Pat. Nos. 5,846,709 and 5,843,650, ligase chain reaction, nucleic acid sequencing and the like.
Although PCR provides numerous advantages in research, the use of thermal cycling on a large scale in clinical laboratories is not widespread. This is largely due to the fact that complex and cumbersome steps are required to prepare nucleic acid samples for analysis. Such steps when effected for a large number of samples, as is typical in diagnostics, are time consuming and may lead to the generation of errors and contamination and/or expose workers to possible infection when effected manually. Furthermore, since the products of such PCR reactions must be analyzed to yield diagnostic results, transfer of the samples to an analytic instrument or in turn, real time analysis must be effected in an automatic manner.
To overcome some of these limitations, the use of an automated sample preparation coupled to thermal cyclers for large scale PCR reactions is practiced.
For example, Beckman Instruments, Inc. (Fullerton, Calif.) provides the Biomek.RTM. 2000 automated pipetting apparatus, that can automate the sample preparation steps for PCR or DNA sequencing reactions in a 96 well microtiter plate using a group of eight pipetting tips. Trays containing reagents or samples are arranged for sequential liquid transfer functions.
Another pipette robot, the Qiagen BioRobot.TM. 9600 (Qiagen Inc., Chatsworth, Calif.) can prepare 96 bacterial minipreps in 2 hours. These robots all use a cooling plate to keep the reagents and samples at controlled temperatures (usually 4.degree. C.) during sample preparation.
The reagent trays prepared in apparatuses of this type are then generally transferred typically automatically to a separate instrument for purposes of thermal cycling.
For example, the RoboCycler.TM. Gradient 96 System (Stratagene, Inc.) has 4 different temperature blocks and a lifter that moves a tray of up to 96 tubes from block to block in sequence. In this way, the apparatus cycles reaction mixtures through a series of preset temperatures as appropriate for amplification or sequencing reactions.
The Vistra.TM. DNA Labstation 625 (Molecular Dynamics, Sunnyvale, Calif.) is a pipette robot that can prepare bacterial mini-preps and PCR and DNA sequencing reactions in a 96 well microtiter plate. The Labstation 625 has an integrated Peltier-block thermocycler for thermal-cycling steps. Using this apparatus, a technician can prepare a sequencing experiment in about 10-15 minutes, and then start the thermocycling procedure. This apparatus uses tubes, and places a layer of oil on top of the reactions to reduce loss of sample during heating.
While there are many advantages to combining sample preparation and thermal cycling into a single apparatus, a further limitation which is not addressed by the above, is the automatic provision of the end products from PCR reaction to appropriate analysis devices, or alternatively analysis of these products during the course of the PCR reaction.
To partially overcome this problem, U.S. Pat. No. 5,897,842 describes an apparatus which automates the large number of pipetting steps and the thermocycling steps involved in preparing a nucleic acid sample while, at the same time, it is designed to automatically provide the resultant end products to analytic devices for further analysis.
Although the above mentioned apparatus provides major advantages over the above described art, it still suffers from several limitations.
To effect such automation the apparatus described in U.S. Pat. No. 5,897,842 utilizes flow-through reaction vessels, such as capillary tubes, for the preparation and thermal cycling of reaction mixtures. In order to prevent loss of the reaction mixture from the vessels during heating, the thermal cycling apparatus provides a formable seal for transiently sealing the distal end of each reaction vessel while positive pressure transiently seals the proximal end of the reaction vessel following the application of the formable seal to the distal end thereof.
As further described in the above patent, both generation of the positive pressure and sample drawing into the reaction vessels are effected by a single pump. Thus, to prevent cross contamination between the samples an appropriate fluid barrier, which can be provided within the proximal end of the reaction vessel must be utilized. Such a barrier is either described nor mentioned by U.S. Pat. No. 5,897,842, and as such, his apparatus is particularly prone to cross contamination of samples.
There is thus a widely recognized need for, and it would be highly advantageous to have, an apparatus and method for effecting automated nucleic acid reactions, such as PCR, while at the same time enabling analysis of the resultant products either during (real-time) or following the reaction, and yet be devoid of the above limitation.